A commitment has been made in the USA to vaccinate large numbers of persons against anthrax. However, limitations of the current anthrax vaccine are widely acknowledged, and efforts are underway to improve the vaccine formulation. We will create biological markers and predictors of the response to anthrax vaccination, with the long-term clinical goal of designing safer and more effective vaccines. To achieve this objective, we will develop novel human class II MHC tetramers which identify antigen-specific CD4+ T cells specific for bacillus anthrax Protective Antigen (BAPA). Quantitation and phenotyping of the tetramer-positive T cells following anthrax vaccination will be evaluated as an indicator of cellular immunity. The Specific Aims are: Aim 1. To identify immunodominant epitopes of bacillus anthrax Protective Antigen (BAPA) based on HLA class II binding and recognized by CD4 T cells which are capable of eliciting robust immunity. Aim 2. To use soluble HLA-BAPA tetramers to quantify the CD4 T cell response to anthrax vaccination. Tetramers will be produced and evaluated which encompass most of the prevalent human class II haplotypes, based on selection of peptides corresponding to T cell recognition of a restricted set of BAPA epitopes. MHC-peptide binding assays, tetramer production and testing methods, and epitope scanning technologies for this project are all validated, and preliminary data using transgenic murine surrogates for the T cell response to BAPA demonstrate successful detection of the responding antigen-specific population following vaccination.